The present invention relates to three dimensional structures of Fc receptors (FcR), including crystalline Fcxcex3RIIa, crystalline Fcxcex5RI, three dimensional coordinates of Fcxcex3RIIa protein, a three dimensional structure of Fcxcex3RIIa, three dimensional structures of FcR, and particularly Fcxcex5RI and Fcxcex3RIIIb, derived from the structure of Fcxcex3RIIa, models thereof, and uses of such structures and models.
Fc receptors (FcR) are a family of highly related receptors that are specific for the Fc portion of immunoglobulin (Ig). These receptors have major roles in normal immunity and resistance to infection and provide the humoral immune system with a cellular effector arm. Receptors have been defined for each of the immunoglobulin classes and as such are defined by the class of Ig of which they bind (i.e. Fc gamma receptor (Fcxcex3R) bind gamma immunoglobulin (IgG), Fc epsilon receptor (Fcxcex5R) bind epsilon immunoglobulin (IgE), Fc alpha receptor (Fcxcex1R) bind alpha immunoglobulin (IgA)). Among the Fcxcex3R receptors, three subfamily members have been defined; Fcxcex3RI, which is a high a affinity receptor for IgG; Fcxcex3RII, which are low affinity receptors for IgG that avidly bind to aggregates immune complexes; and Fcxcex3RIII, which are low affinity receptors that bind to immune complexes. These receptors are highly related structurally but perform different functions. The structure and function of Fcxcex3RII is of interest because of its interaction with immune complexes and its association with disease.
Fcxcex3R are expressed on most hematopoietic cells, and through the binding of IgG play a key role in homeostasis of the immune system and host protection against infection. Fcxcex3RII is a low affinity receptor for IgG that essentially binds only to IgG immune complexes and is expressed on a variety of cell types including, for example monocytes, macrophages, neutrophils, eosinophils, platelets and B lymphocytes. Fcxcex3RII is involved in various immune and inflammatory responses including antibody-dependent cell-mediated cytotoxicity, clearance of immune complexes, release of inflammatory mediators and regulation of antibody production. The binding of IgG to an Fcxcex3R can lead to disease indications that involve regulation by Fcxcex3R. For example, the autoimmune disease thrombocytopenia purpura involves tissue (platelet) damage resulting from Fcxcex3R-dependent IgG immune complex activation of platelets or their destruction by Fcxcex3R+ phagocytes. In addition, various inflammatory disease are known to involve IgG immune complexes (e.g. rheumatoid arthritis, systemic lupus erythematosus), including type II and type III hypersensitivity reactions. Type II and type III hypersensitivity reactions are mediated by IgG, which can activate either complement-mediated or phagocytic effector mechanisms, leading to tissue damage.
The elucidation of the protein structure of Fcxcex3RIIa, Fcxcex5RI, or indeed any FcR is of importance in the formulation of therapeutic and diagnostic reagents for disease management. Until the discovery of the present invention, the structure and resulting mechanism by which Fcxcex3RIIa regulates immune responses was unknown. Thus, despite the general multifunctional role of Fcxcex3RIIa, development of useful reagents for treatment or diagnosis of disease was hindered by lack of structural information of the receptor. The linear nucleic acid and amino acid sequence of Fcxcex3RIIa have been previously reported (Hibbs et al. Proc. Natl. Acad. Sci. USA, vol. 85, pp. 2240-2244, 1988). Mutagenesis studies to identify regions of human Fcxcex3RIIa (Hulett et al., Eur. J Immunol., vol. 23, pp. 40-645, 1993; Hulett et al., J. Biol. Chem., vol. 69, pp. 15287-15293 1994; and Hulett et al., J. Biol. Chem., vol. 270, pp. 21188-21194, 1995), human Fcxcex3RIIIb (Hibbs et al., J. Immunol., vol. 152, p. 4466, 1994; and Tamm et al., J. Biol. Chem., vol. 271, p. 3659, 1996) and mouse Fcxcex3RI (Hulett et al., J. Immunol., vol. 148, pp. 1863-1868, 1991) have defined important regions of IgG binding to the Fcxcex3R. Information based on linear sequences, however, cannot accurately predict three dimensional structure of the protein and its functional domains. Huber et al. (J. Mol. Biol., vol. 230, pp. 1077-1083, 1993) have described crystal formation of neonatal rat Fc receptor protein (FcRn). Burmeister et al. (Nature, vol. 372, pp. 336-343, 1994; and Nature, vol. 372, pp. 379-383, 1994) have described the structure of FcRn crystals. FcRn, however, is closely related to major histocompatability protein complex and not related to the leukocyte Fcxcex3R family by function or structure. Thus, the protein structure of FcRn is not predictive of the FcR structure of the present invention.
Fcxcex5R are expressed on mast cells, and through the binding of IgE, trigger an inflammatory immune response which is primarily due to the release of inflammatory mediators upon degranulation of the mast cell (e.g., histamine and serotonin). Release of these mediators causes localized vascular permeability and increase in fluids in the local tissues, including an influx of polymorphonuclear cells into the site. Thus, binding of IgE to an Fcxcex5RI can lead to disease indications that involve discharge of fluids from the gut and increased mucus secretion and bronchial contraction, such indications typically being associated with diseases involving allergic inflammation. Therefore, the elucidation of protein structure of Fcxcex5RI is of importance in the formulation of therapeutic and diagnostic reagents for disease management, and in particular, for the management of diseases related to allergic inflammation and other Th2-based immune responses. As for the Fcxcex3R described above, the linear nucleic acid and amino acid sequences of human Fcxcex5RI have been previously reported (Kochan et al., 1998, Nuc. Acid. Res. 16:3584). Until the discovery of the present invention, however, the structure and resulting mechanism by which Fcxcex5R regulates immune responses was unknown. Thus, despite the knowledge of the general action of Fcxcex5RI, the development of useful reagents for treatment or diagnosis of disease, such as diseases associated with allergic inflammation, was hindered by lack of structural information of the receptor.
Therefore, there is a need in the art to elucidate the three dimensional structures and models of the Fc receptors, and to use such structures and models in therapeutic strategies, such as drug design.
The present invention relates to crystalline Fcxcex3RIIa and crystalline Fcxcex5RI, three dimensional coordinates of Fcxcex3RIIa protein, the three dimensional structure of Fcxcex3RIIa, three dimensional structures and models of Fc receptors (FcR) derived from the structure of Fcxcex3RIIa, including Fcxcex5RI and Fcxcex3RIIIb, and uses of such structures and models. Obtaining such crystals is an unexpected result. It is well known in the protein crystallographic art that obtaining crystals of quality sufficient for determining the structure of a protein is unpredictable. In particular, obtaining crystals of quality sufficient for determining the three dimensional (3-D) structure of Fcxcex3RIIa has not been achievable until the crystallization of Fcxcex3RIIa as disclosed in the present application. As such, determination of the three dimensional structure of Fcxcex3RIIa has not been possible until the discovery of the present invention. Additionally, until the discovery of the present invention, derivation of the three dimensional structure and models of other Fc receptor (FcR) proteins has not been possible. The present inventors are also the first to define the three dimensional structure and provide three dimensional models for drug design for Fcxcex5RI and Fcxcex3RIIIb.
Accordingly, one object of the present invention is to provide crystals of sufficient quality to obtain a determination of the three dimensional structure of Fcxcex3RIIa to high resolution, preferably to the resolution of about 1.8 angstrom. The present invention also includes methods for producing crystalline Fcxcex3RIIa.
Yet another object of the present invention is to provide crystals of Fcxcex5RI protein, preferably of sufficient quality to obtain a determination of the three dimensional structure of Fcxcex5RI to high resolution. The present invention also includes methods for producing crystalline Fcxcex5RI.
The value of the crystals of Fcxcex3RIIa and Fcxcex5RI extends beyond merely being able to obtain such crystals. The knowledge obtained concerning the Fcxcex3RIIa crystal structure, for example, has been used by the present inventors to define the heretofore unknown tertiary structure of the Fcxcex3RIIa protein, to model and derive atomic coordinates for the heretofore unknown tertiary structure of the Fcxcex5RI protein and the heretofore unknown tertiary structure of the Fcxcex3RIIIb protein, and can be additionally used to model the heretofore unknown tertiary structure of other FcR proteins having substantially related linear amino acid sequence, such as for other members of the Fcxcex3R protein family and the Fcxcex1RI protein. There are three members of the Fcxcex3R family of proteins, Fcxcex3RI, Fcxcex3RII and Fcxcex3RIII, all of which act as immunoregulatory molecules and all of which bind to IgG. Comparison of nucleic acid and amino acid sequences of the Fcxcex3R family of receptors indicates that the receptors are highly homologous. In addition, each member of the Fcxcex3R family of receptors belongs to the Ig super family of molecules, an assignment based on well established criteria (Hulett et al. 1994, ibid.). Moreover, Fcxcex3RII, Fcxcex3RIII, Fcxcex5RI and Fcxcex1RI each contain Ig-like domains, indicating the similarity between these receptors. Fcxcex3RI contains three Ig-like domains. The first and second domains, however, of Fcxcex3RI are substantially homologous to the Ig-like domains of Fcxcex3RII, Fcxcex3RIII, Fcxcex5RI and Fcxcex1RI. Current methods of tertiary structure determination that do not rely on x-ray diffraction techniques and thus do not require crystallization of the protein (e.g., computer modeling and nuclear magnetic resonance techniques) enable derivation and refinement of models of other Fcxcex3R proteins, Fcxcex5RI and Fcxcex1RI protein, extrapolated from a three dimensional structure of Fcxcex3RIIa protein. Thus, knowledge of the three dimensional structure of Fcxcex3RIIa protein has provided a starting point for investigation into the structure of all of these proteins.
Accordingly, a second object of the present invention is to provide information regarding the structure of Fcxcex3RIIa protein and models, atomic coordinates and derived three dimensional structures of other members of the Fcxcex3R family of proteins, Fcxcex5RI and Fcxcex1RI protein.
The knowledge of the three dimensional structure of Fcxcex3RIIa and models of other FcR provides a means for designing and producing compounds that regulate immune function and inflammation in an animal, including humans (i.e., structure based drug design). For example, chemical compounds can be designed to block binding of immunoglobulin to an Fc receptor protein using various computer programs and models.
Another embodiment of the present invention is to provide a three dimensional computer image of the three dimensional structure of an FcR.
Another embodiment of the present invention is to provide a computer-readable medium encoded with a set of three dimensional coordinates selected from the group of the three dimensional coordinates represented in Table 1, the three dimensional coordinates represented in Table 2, the three dimensional coordinates represented in Table 3, the three dimensional coordinates represented in Table 4, and the three dimensional coordinates represented in Table 5, wherein, using a graphical display software program, the three dimensional coordinates create an electronic file that can be visualized on a computer capable of representing said electronic file as a three dimensional image.
Accordingly, a third object of the present invention is to provide methods for using a three dimensional structure of FcR, such as Fcxcex3RIIa, and structures, coordinates and models derived using such structure, for designing reagents for the treatment and diagnosis of disease, such as by binding to or mimicking the action of FcR protein, binding to or mimicking the action of an immunoglobulin (Ig), disrupting cellular signal transduction through an FcR protein by, for example, preventing dimerization of two FcR proteins, or enhancing cellular signal transduction or binding to an FcR by, for example, enhancing dimerization of two FcR proteins.
The knowledge of the three dimensional structure of FcR also provides a means for designing proteins that have altered beneficial functions by analyzing the structure and interactions between individual amino acids of the protein. For example, therapeutic proteins having improved binding to Ig or immune complexes of Ig can be designed to be used as therapeutic compounds to prevent immune complex binding to cells or enhance biological responses such as cellular signal transduction upon binding of FcR to Ig or complexes thereof. Thus recombinant soluble FcR engineered to contain improvements can be produced on the basis of the knowledge of the three dimensional structure.
Accordingly, a fourth object of the present invention is to provide for an extrapolation of the three dimensional structure of FcR to create recombinant protein having altered biological activity.
One embodiment of the present invention is a model of an FcR protein, wherein the model represents the three dimensional structure of FcR protein, in which the structure substantially conforms to the atomic coordinates represented by Table 1. Other embodiments of the present invention are the three dimensional structure of an Fcxcex3RIIa protein which substantially conforms to the atomic coordinates represented by Table 1; the three dimensional structure of a dimeric Fcxcex3RIIa protein which substantially conforms to the atomic coordinates represented by Table 2; the three dimensional structure of a monomeric Fcxcex5RI protein which substantially conforms to the atomic coordinates represented by Table 3; the three dimensional structure of a dimeric Fcxcex5RI protein which substantially conforms to the atomic coordinates represented by Table 4; the three dimensional structure of a dimeric Fcxcex3RIIIb protein which substantially conforms to the atomic coordinates represented by Table 5 and models representing such structures. Further embodiments of the present invention relate to a set of three dimensional coordinates of an Fcxcex3RIIa protein, wherein said coordinates are represented in Table 1; a set of three dimensional coordinates of a dimeric Fcxcex3RIIa protein, wherein said coordinates are represented in Table 2; a set of three dimensional coordinates of an Fcxcex5RI protein, wherein said coordinates are represented in Table 3; a set of three dimensional coordinates of an Fcxcex5RI protein, wherein said coordinates are represented in Table 4; and a set of three dimensional coordinates of Fcxcex3RIIIb, wherein said coordinates are represented in Table 5. The present invention also includes methods to use such structures including structure based drug design and methods to derive models and images of target FcR structures.
Another embodiment of the present invention is a composition comprising Fcxcex3RIIa protein in a crystalline form. Yet another embodiment of the present invention is a composition comprising Fcxcex5RI protein in a crystalline form.
Yet another embodiment of the present invention is a method for producing crystals of Fcxcex3RIIa, comprising combining Fcxcex3RIIa protein with a mother liquor buffer selected from the group consisting of an acetate salt buffer and a sulphate buffer, and inducing crystal formation to produce said Fcxcex3RIIa crystals.
The present invention also includes a method for producing crystals of Fcxcex5RI, comprising combining Fcxcex5RI protein with a mother liquor buffer selected from the group consisting of an acetate salt buffer, a sodium cacodylate buffer and a sodium citrate buffer, and inducing crystal formation to produce said Fcxcex5RI crystals.
The present invention also includes a therapeutic composition that, when administered to an animal, reduces IgG-mediated tissue damage, said therapeutic composition comprising an inhibitory compound that inhibits the activity of an Fcxcex3RIIa protein, said inhibitory compound being identified by the method comprising: (a) providing a three dimensional structure of an Fcxcex3RIIa protein; (b) using said three dimensional structure to design a chemical compound selected from the group consisting of a compound that inhibits binding of Fcxcex3RIIa protein to IgG, a compound that substantially mimics the three dimensional structure of Fcxcex3RIIa protein and a compound that inhibits binding of Fcxcex3RIIa protein with a molecule that stimulates cellular signal transduction through an Fcxcex3RIIa protein; (c) chemically synthesizing said chemical compound; and (d) evaluating the ability of said synthesized chemical compound to reduce IgG-mediated tissue damage.
Another embodiment of the present invention is a therapeutic composition that is capable of stimulating an IgG humoral immune response in an animal. Yet another embodiment of the present invention is a therapeutic composition that improves the therapeutic affects of an antibody that is administered to an animal to treat, by opsonization or Fcxcex3R-dependent effector functions (e.g. antibody-dependent Fcxcex3R-medicated cytotoxicity, phagocytosis or release of cellular mediators), a particular disease, including, but not limited to, cancer or infectious disease (e.g. oral infections such as HIV, herpes, bacterial infections, yeast infections or parasite infections). Such a therapeutic composition includes one or more stimulatory compounds that have increased binding to IgG, enhance binding of IgG to Fcxcex3R, enhance dimer formation of an Fcxcex3R and/or enhance signal transduction through the Fcxcex3R. Also included in the present invention is a method to stimulate a humoral immune response. The method includes the step of administering to an animal a therapeutic composition of the present invention.
The present invention also includes a therapeutic composition that, when administered to an animal, reduces IgG-mediated tissue damage, said therapeutic composition comprising an inhibitory compound that inhibits the activity of an Fcxcex3RIIIb protein, said inhibitory compound being identified by the method comprising: (a) providing a three dimensional structure of an Fcxcex3RIIIb protein; (b) using said three dimensional structure to design a chemical compound selected from the group consisting of a compound that inhibits binding of Fcxcex3RIIIb protein to IgG, a compound that substantially mimics the three dimensional structure of Fcxcex3RIIIb protein and a compound that inhibits binding of Fcxcex3RIIIb protein with a molecule that stimulates cellular signal transduction through an Fcxcex3RIIIb protein; (c) chemically synthesizing said chemical compound; and (d) evaluating the ability of said synthesized chemical compound to reduce IgG-mediated tissue damage.
One embodiment of the present invention is a therapeutic composition that is capable of reducing IgE-mediated responses. Such therapeutic compositions are capable of reducing IgE-mediated responses resulting from IgE-mediated hypersensitivity, IgE-mediated release of inflammatory modulators or other biological mechanisms involved in IgE-mediated recruitment of inflammatory cells that involves Fcxcex5R protein. Such a therapeutic composition of the present invention can: (1) inhibit (i.e., prevent, block) binding of Fcxcex5R protein on a cell having an Fcxcex5R protein (e.g., mast cells) to an IgE immune complex by interfering with the IgE binding site of an Fcxcex5R protein; (2) inhibit precipitation of IgE or IgE immune complexes (i.e., prevent Fc:Fc interactions between two IgE); (3) inhibit immunoglobulin-mediated cellular signal transduction by interfering with the binding of an IgE to a cell surface receptor; and (4) inhibit Fcxcex5R-mediated cellular signal transduction by interfering with the binding of a cell signal inducing molecule (i.e., a molecule that induces cellular signal transduction through an Fcxcex5R protein) to an Fcxcex5R protein. Such therapeutic compositions include one or more inhibitory compounds that inhibit binding of IgE to Fcxcex5R protein, IgE to IgE, IgE to a cell surface receptor, or a cell signal inducing molecule to Fcxcex5R protein. Also included in the present invention are methods to reduce IgE-mediated responses, such as IgE-mediated inflammation.
Another embodiment of the present invention is a therapeutic composition that is capable of stimulating a IgE humoral immune response in an animal. Yet another embodiment of the present invention is a therapeutic composition that improves the therapeutic affects of an antibody that is administered to an animal to treat, by opsonization or Fcxcex5R-dependent effector functions (e.g. phagocytosis or release of cellular mediators), a particular disease. Such a therapeutic composition includes one or more stimulatory compounds that have increased binding to IgE, enhance binding of IgE to Fcxcex5RI, enhance dimer formation of Fcxcex5RI and/or otherwise enhance signal transduction through the Fcxcex5RI. Also included in the present invention is a method to stimulate a humoral immune response. The method includes the step of administering to an animal a therapeutic composition of the present invention.
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